Biosensors are electrodes which employ enzymes or other biologically active materials as highly selective catalysts. Early biosensors contained an enzyme in aqueous solution which was held in proximity to a sensing electrode by a membrane which did not contain a significant amount of the enzyme. The membranes of these biosensors served primarily as a physical barrier which, by virtue of their small pore size, prevented the enzyme from migrating into the bulk solution. The membrane also served to keep large protein molecules from entering the aqueous enzyme solution and interfering with planned reactions. These biosensors were comparatively large and their reaction times were slow by modern standards.
One improved type of biosensor held an enzyme trapped between two or more individual membranes. The enzyme could be dispersed in an adhesive which held the membranes in place, especially when the enzyme was glucose oxidase and the adhesive was glutaraldehyde. The restraining membranes did not contain a significant amount of enzyme.
Another type of biosensor contains enzymes immobilized directly within a permeable or semipermeable carrier. The enzyme may be physically encapsulated within the membrane in the form of fine droplets dispersed in a polymer. Alternately, the enzyme may be covalently bonded to functional polar groups which are attached to a synthetic polymer membrane. Functional groups include covalent binding groups, ionic binding groups, and hydrophobic groups. The immobilized enzyme membranes contain micropores which facilitate diffusion of target reagents and reaction products through the membrane.
Covalent bonds for immobilizing proteins are formed by linking amino or carboxyl groups, which are present in every enzyme, with polar functional groups which may be present on the carrier membrane. The functional groups can be derived from components normally present in the materials that forms a carrier substrate or the functional groups can be added to the carrier substrate. Suitable functional groups include carboxyl groups, amino groups, sulfonic acid groups, imino groups, thio groups, hydroxyl groups, pyridyl groups, and phosphoryl groups. The functional groups may be pre-activated by chemical treatment to enhance their ability to join with the amino or carboxyl groups present in the enzyme molecules.
As biosensor technology progressed, laminated membranes were produced which included an enzyme membrane which carried immobilized proteins and other membranes. The other membranes are located on one or both sides of the enzyme membrane and serve to impede the movement of undesirable species. For example, protecting membranes having relatively large pores are included in laminated membranes between an analyte solution and an enzyme membrane in that position, the protecting membranes prevent high molecular weight species from becoming adsorbed upon and fouling the surface of the enzyme membrane. Similarly, blocking membranes with relatively smaller pores were included between the enzyme membrane and a sensing electrode to prevent low molecular weight species, such as ascorbic acid and paracetamol from interfering with electrical detection and measurement as the sensing electrode. Some of the laminated membranes are prepared by the technique of spin casting successive layers of polymeric material.
The laminated membranes are not entirely satisfactory, however. They are expensive and time-consuming to prepare. Achieving a uniform distribution of enzyme attachment within the enzyme membrane is problematic. In addition, the laminated membranes frequently leak at their point of attachment to a sensor device and an analyte solution is then able to bypass the enzyme. Laminated membranes can also separate during operation.
Accordingly, improved laminated membranes and improved methods of manufacturing have been sought, as well as substitutes. One substitute is a blocking layer deposited directly upon the sensing electrodes. Such blocking layers are produced by polymerizing compounds directly on the electrodes, such as diaminobenzene and dihydroxybenezene copolymers. Intimate contract is achieved between the sensing electrode and the blocking membrane, but the quality of devices produced by this technique is often inconsistent.
Over the last two decades, evolution in biosensor design has been stimulated by a demand for smaller, faster, and more reliable leak-proof sensors which exhibit high sensitivity. Such sensors are especially useful for constructing arrays which contain many sensors. The demand for small biosensors with faster response and recovery times appeared to be at odds with the requirements of leak-proof reliability and high sensitivity. Generally, the route to faster response times is by incorporation of more sophisticated, less diffusion-resistant membranes, and through improved immobilization techniques which uniformly distribute the enzyme in well-defined layers without exposing the enzyme to conditions which might denature it.
In contrast, the use of sophisticated membranes and advanced immobilization techniques places a premium on the integrity of a seal between the enzyme membrane and the housing. Previously, the seal had been made using clamps, mechanical fasteners, or adhesives, and was a frequent source of leakage. Leakage around the enzyme would reduce the sensitivity of the biosensor device because reagents which had not been converted would not register at the electrode. Of course, the leakage bypass problem is most critical for small biosensor devices where the deleterious effect of any given size leak becomes relatively more important. When the seal leaks, the time required to rinse out a sample or calibrant solution is extended. Avoiding leaks is especially important in the case of systems with multiple sensors.